It has long been established that in the absence of LH, as occurs in hypophysectomized animals, Leydig cell development and function are drastically altered. However, it has recently been shown that even when LH is present, Leydig cells produce far less testosterone when macrophages are depleted from the testis. Similarly, when macrophages are depleted from the testis of immature animals, Leydig cells fail to develop properly. Thus testicular macrophages, like LH, are essential for normal function of Leydig cells. We have partially purified and characterized a lipophilic factor secreted by macrophages that stimulates testosterone production by Leydig cells. We hypothesize that this factor mediates some of the actions that macrophages exert on Leydig cells that have been described in these macrophage-depletion studies, including a role in Leydig cell maturation, stimulation of testosterone production, and involvement in maintaining LH- responsiveness. Thus this factor has the potential to be of great physiological significance. In Aim 1 we propose to use mass spectroscopy, nuclear magnetic resonance, and other physico-chemical approaches to identify the chemical structure of this factor. A CoPI and a collaborator have been added to the proposal as recommended who have expertise in this area. In Aim 2, we will continue studies concerning the mechanism of action of the factor. We have found that the factor elicits a greater response than does LH/hCG, does not require new synthesis of StAR, does not act directly on mitochondria, is not itself a substrate for steroidogenesis, and exerts its influence proximal to cholesterol sidechain cleavage, all indicating that its mode of action is very different than that of LH. We now propose to determine if the factor activates protein kinase A and/or C, causes changes in calcium metabolism, and/or changes in intracellular levels of cAMP. In Aim 3, we will determine if the factor is produced by testicular macrophages from immature animals. If so, we will then test its ability to induce maturation of postnatal Leydig cells. Finally, we will determine if the factor can restore Leydig cell function (testosterone production and ability to respond to LH) in macrophage- depleted animals. These gain-of-function studies will determine the physiological significance of this factor, and if positive results are obtained, new opportunities to treat infertility and/or create new male contraceptives may emerge, since testicular macrophages are present in high numbers in humans as they are in rodents.